Volume 30, Number 9—September 2024
Dispatch
Cocirculation of Genetically Distinct Highly Pathogenic Avian Influenza H5N5 and H5N1 Viruses in Crows, Hokkaido, Japan
Abstract
We isolated highly pathogenic avian influenza (HPAI) H5N5 and H5N1 viruses from crows in Hokkaido, Japan, during winter 2023–24. They shared genetic similarity with HPAI H5N5 viruses from northern Europe but differed from those in Asia. Continuous monitoring and rapid information sharing between countries are needed to prevent HPAI virus transmission.
H5 highly pathogenic avian influenza viruses (HPAIVs) of the A/goose/Guangdong/1/1996 lineage have diversified into multiple clades, threatening wild birds and poultry worldwide. Clade 2.3.4.4b HPAIVs have been consistently isolated in Asia and Europe since 2016 (1–3) and expanded further to North America in late 2021 (4). The global circulation of H5 HPAIVs over a relatively short time highlights the pivotal role of migratory birds in virus dissemination (5). H5 HPAIVs in clade 2.3.4.4 frequently acquire the neuraminidase (NA) gene from locally circulating low pathogenicity avian influenza viruses (LPAIVs), which often infect waterfowl, leading to the generation of novel H5Nx reassortant viruses, such as H5N2, H5N6, and H5N8 (6).
During the winter seasons 2021–22 and 2022–23, Hokkaido, located in the northernmost part of Japan, experienced HPAIV outbreaks driven by bird migration that substantially affected poultry and other resident birds. Those viruses clustered in the group 2 (G2) d subgroup within clade 2.3.4.4.b, which has multiple subgroups, G2a–e, and shared a common ancestor with HPAIVs detected in Europe in late 2020 (7). HPAIV subgroup G2d might have undergone intercontinental transmission from Europe to Japan (8,9). During winter 2023–24, H5N5 HPAIVs were detected in a crow flock in Hokkaido, and further monitoring revealed cocirculation of 2 distinct viruses in the crow population. We investigated the genetic origin and antigenicity of H5N5 HPAIVs isolated in Hokkaido.
We conducted passive surveillance of HPAIV infections in wild birds in a public garden in Sapporo, the prefectural capital of Hokkaido, Japan; ≈2,000 crows flock together during winter and are observed by garden staff. We isolated viruses from tracheal and cloacal swab samples collected from dead crows in the garden by inoculating 10-day-old embryonated eggs; we confirmed results by using reverse transcription PCR (Appendix). On November 23 and 24, 2023, we isolated H5N1 HPAIVs from 2 dead large-billed crows (Corvus macrorhynchos), designated as A/large-billed crow/Hokkaido/B067/2023 (H5N1) and A/large-billed crow/Hokkaido/B068/2023 (H5N1). The hemagglutinin (HA) gene sequences from those 2 H5N1 HPAIVs indicated they clustered with the G2d subgroup of HPAIVs found in Hokkaido during the winter seasons 2021–22 and 2022–23. In contrast, HA genes of 3 H5 HPAIVs isolated from dead crows on January 8–11, 2024, were closely related to the G2a subgroup of H5N5 HPAIVs found in northern Europe and North America. Subsequent whole-genome sequencing analysis of the 3 G2a HPAIVs confirmed their subtype was H5N5; we named them A/large-billed crow/Hokkaido/B073/2024 (H5N5), A/large-billed crow/Hokkaido/B074/2024 (H5N5), and A/crow/Hokkaido/B075/2024 (H5N5) (Table 1).
We phylogenetically analyzed virus isolates along with reference sequences obtained from GISAID (https://www.gisaid.org); the HA genes of H5N5 HPAIVs isolated in Hokkaido diverged considerably from HPAIVs isolated in Japan during winter 2020–21 (10), forming a distinct branch within the G2a subgroup (Figure). In addition, the other gene segments of H5N5 HPAIVs from Hokkaido were genetically distant from those in HPAIV strains isolated in Japan during winter 2021–22 (Appendix Figures 1–6). BLAST (https://blast.ncbi.nlm.nih.gov) analysis of sequences from GISAID revealed that all 8 gene segments of H5N5 HPAIVs from Hokkaido were very close (genetic similarity >99%) to H5N5 HPAIVs detected in northern Europe since 2022, in contrast to those from North America (Table 2), suggesting a low possibility of virus transmission from North America. H5N5 HPAIVs from Hokkaido shared a common ancestor with H5N5 HPAIV from Europe assigned the genotype EA-2021-I by the European Food Safety Authority (11). Parent strains of H5N5 HPAIVs from Europe, represented by A/swan/Rostov/2299-2/2020 (H5N5), were proposed to originate in western Russia during autumn 2020. Those viruses underwent genetic evolution via reassortment events involving H5N8 HPAIVs circulating in Europe since 2018 (12) and the N5 NA gene derived from concurrently circulating LPAIVs (13). H5N5 HPAIVs reported in northern Europe during 2022–2023 exhibited specific genetic differences compared with H5N5 HPAIVs detected in Europe during autumn 2020, particularly in the N5 NA gene. Those differences included a 66-bp nucleotide deletion within the N5 NA gene, which we also observed in the H5N5 HPAIVs from Hokkaido. Truncation of the NA stalk has been attributed to the adaptation of those viruses from wild birds to Galliformes spp. birds (14). However, most H5N5 HPAIV infections in Europe were detected in wild birds, and no cases have been detected in Galliformes spp. birds since 2022 (15). Further investigation is needed to clarify whether NA stalk truncation affects pathogenesis of H5N5 HPAIVs.
During winter 2023–24, we confirmed H5N5 HPAIV infections in wild birds, especially in crows, in Erimo (December 19, 2023, in south-central Hokkaido) and in Kushiro (January 18, 2024, in eastern Hokkaido); we also confirmed infection in a peregrine falcon (Falco peregrinus) in Tamana, Kumamoto Prefecture, Kyushu Island, on January 16, 2024. We classified the isolate from Tamana, A/peregrine falcon/Kumamoto/4301C001/2024 (H5N5), into the G2d subgroup according to its HA gene sequence, whereas its NA gene sequence was similar to that of LPAIVs isolated in East Asia (Table 2). Although this combination had not been observed in Japan, reassortment events between the HPAIV H5N1 G2d subgroup and LPAIVs have been documented (9). We detected H5N5 HPAIVs in Hokkaido in January 2024; a total of 85 crows were found dead in the Sapporo garden, 80 of which we diagnosed as HPAIV positive by the end of April. No HPAIVs were detected in birds within the garden after April 2024. The continuous detection of H5N5 HPAIVs in the Sapporo garden during January–April without unusual deaths of birds other than crows and multiple isolations of H5N5 HPAIVs in other areas of Hokkaido suggest the potential for widespread dissemination of H5N5 HPAIVs within the Hokkaido region.
H5N1 G2d HPAIVs persisted in crows residing in the Sapporo garden even after the introduction of H5N5 G2a viruses, indicating concurrent circulation of genetically distinct viruses within a single crow population. Indeed, the average nanopore sequencing coverage for A/large-billed crow/Hokkaido/B080/2024 (H5N1) was 5497.4 reads for the N1 NA gene (G2d subgroup) and 1943.7 reads for the N5 NA gene (G2a subgroup) (Appendix Table 1). This observation suggests single hosts were co-infected with 2 viruses and reassortment occurred between viruses originating from geographically distant areas. Antigenic characterization of H5N5 HPAIVs suggested that the antigenicity of A/large-billed crow/Hokkaido/B073/2024 (H5N5) was close (2–4-fold differences in hemagglutination inhibition titers) to that of other H5 HPAIVs in the G2d subgroup (Table 3) despite their genetic diversity (Appendix Table 2).
We found that H5N5 HPAIVs consisting of unique gene constellations were likely introduced into Japan through a step-by-step bird migration through northern Eurasia. We confirmed the cocirculation of 2 genetically distinct viruses in a single flock of crows. The presence of H5N5 HPAIV infections in waterfowl in Japan is relatively unknown, and the lack of reports from neighboring countries on the presence of H5N5 HPAIVs from Europe has hampered the reconstruction of this genotype’s spread to eastern Asia. Continuous monitoring and rapid information sharing between countries are needed to determine the global dynamics of HPAIVs and prevent their spread.
Mr. Hew is a PhD candidate at Hokkaido University, Sapporo, Japan. His primary research focuses on the molecular diagnosis and epidemiology of avian influenza viruses.
Acknowledgments
We thank Mayumi Endo and Fumihito Takaya for their technical assistance; Japan Ministry of the Environment and Hokkaido Prefecture for their kind cooperation, and the authors and laboratories that identified and submitted the sequences to the GISAID's EpiFlu database that were used in this study. All GISAID data submitters can be contacted directly via the GISAID website (https://www.gisaid.org).
This work was supported by the Japan Agency for Medical Research and Development (grant no. JP223fa627005). This work was also partially supported by the Japan International Cooperation Agency within the framework of the Science and Technology Research Partnership for Sustainable Development (grant no. JP23jm0110019); Japan Science and Technology Agency’s Support for Pioneering Research Initiated by the Next Generation (grant no. JPMJSP2119); the research project on Regulatory Research Projects for Food Safety, Animal Health, and Plant Protection (grant no. JPJ008617.23812859) funded by the Ministry of Agriculture, Forestry, and Fisheries of Japan; the Doctoral Program for World-Leading Innovative and Smart Education, powered by the Japan Ministry of Education, Culture, Sports, Science and Technology; and the WISE Grant-in-Aid for graduate students, Program for One Health Frontier of the Graduate School of Excellence, Hokkaido University (grant no. PH36210001).
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Cite This ArticleOriginal Publication Date: August 06, 2024
Table of Contents – Volume 30, Number 9—September 2024
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Please use the form below to submit correspondence to the authors or contact them at the following address:
Takahiro Hiono, One Health Research Center, Hokkaido University, North 18, West 9, Kita-ku, Sapporo, Hokkaido 060-0818, Japan
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